In-vitro and in-vivo monitoring of gold(III) ions from intermediate metabolite of sodium aurothiomalate through water-soluble ruthenium (II) complex-based luminescent probe

Article abstract of DOI:10.1016/j.bioorg.2021.104749

Real-time monitoring of drug metabolism in vivo is of great significance to drug development and toxicology research. The purpose of this study is to establish a rapid and visual in vivo detection method for the detection of an intermediate metabolite of the gold (I) drug. Gold (I) drugs such as sodium aurothiomalate (AuTM) have anti-inflammatory effects in the treatment of rheumatoid arthritis. Gold(III) ions (Au³⁺) are the intermediate metabolite of gold medicine, and they are also the leading factor of side effects in the treatment of patients. However, the rapid reduction of Au³⁺ to Au⁺ by thiol proteins in organisms limits the in-depth study of metabolism of gold drugs in vivo. Here we describe a luminescence Au³⁺ probe (RA) based on ruthenium (II) complex for detecting Au³⁺ in vitro and in vivo. RA with large Stokes shift, good water solubility and biocompatibility was successfully applied to detect Au³⁺ in living cells and vivo by luminescence imaging, and to trap the fluctuation of Au³⁺ level produced by gold (I) medicine. More importantly, the luminescent probe was used to the detection of the intermediate metabolites of gold (I) drugs for the first time. Overall, this work offers a new detection tool/method for a deeper study of gold (I) drugs metabolite.

Full text of DOI:10.1016/j.bioorg.2021.104749

Bioorganic Chemistry 110 (2021) 104749
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
In-vitro and in-vivo monitoring of gold(III) ions from intermediate
metabolite of sodium aurothiomalate through water-soluble ruthenium (II)
complex-based luminescent probe
,
Zhenzhen Xiea b, Jia Wenc, Shaowei Sunc, Jing Zhanga, Xiling Denga, Shichao Hand,
,
,*
Lixia Wangd, Bo Zhanga, Chenglin Hongb *, Shiguo Suna
a Key Laboratory of Xinjiang Phytomedicine Resources of Ministry of Education, School of Pharmacy, Shihezi University, Shihezi 832002, China
b Key Laboratory for Green Processing of Chemical Engineering of Xinjiang Bingtuan, School of Chemistry and Chemical Engineering, Shihezi University, Shihezi 832003,
China
c Shaanxi Key Laboratory of Natural Products & Chemical Biology, College of Chemistry & Pharmacy, Northwest Agriculture and Forestry University, Yangling, Shaanxi
712100, China
d Department of Gynaecology and Obstetrics, The Second Affiliated Hospital of Dalian Medical University, Dalian, Liaoning 116027, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Real-time monitoring of drug metabolism in vivo is of great significance to drug development and toxicology
research. The purpose of this study is to establish a rapid and visual in vivo detection method for the detection of
an intermediate metabolite of the gold (I) drug. Gold (I) drugs such as sodium aurothiomalate (AuTM) have anti-
inflammatory effects in the treatment of rheumatoid arthritis. Gold(III) ions (Au3+) are the intermediate
metabolite of gold medicine, and they are also the leading factor of side effects in the treatment of patients.
However, the rapid reduction of Au3+ to Au+ by thiol proteins in organisms limits the in-depth study of meta-
bolism of gold drugs in vivo. Here we describe a luminescence Au3+ probe (RA) based on ruthenium (II) complex
for detecting Au3+ in vitro and in vivo. RA with large Stokes shift, good water solubility and biocompatibility was
successfully applied to detect Au3+ in living cells and vivo by luminescence imaging, and to trap the fluctuation
of Au3+ level produced by gold (I) medicine. More importantly, the luminescent probe was used to the detection
of the intermediate metabolites of gold (I) drugs for the first time. Overall, this work offers a new detection tool/
method for a deeper study of gold (I) drugs metabolite.
Luminescence imaging
Au3+
Gold (I) drug
Ruthenium (II) complex
Large Stokes shift
Water-solubility
1. Introduction
CD4+ T cells susceptible, thereby causing immunotoxicity [6,7].
Although toxicological mechanism of gold drugs has not been fully
Gold (I) drugs, such as sodium aurothiomalate (AuTM), were char-
acterized in 1929 and were clinically used to treat patients with rheu-
matoid arthritis [1]. Gold therapy has attracted numerous research
attention as gold (I) compounds have shown efficacy to retard or even
stop the progress of rheumatoid arthritis, but their use is limited due to
consequential toxic side effects [2,3]. It is believed that phagocytes play
a role in the treatment with gold drugs and the oxidation of gold(I) to
gold(III) (Au3+) ions could occur by Myeloperoxidase(MPO)-H2O2-
halide system in phagocytes [4,5]. Since mononuclear macrophages are
present in many organs and communicate with T cells, the Au3+ pro-
duced by the drug in macrophages can oxidize self-protein, which makes
proven, but experimental evidences indicate their dominants immuno-
toxicity resulting from Au3+. Therefore, there is an urgent need for a
detection method with high sensitivity and selectivity for detecting Au3+
in vivo.
The current research on the detection and mechanism of the inter-
mediate metabolite Au3+ of gold (I) drugs is performed in non-cellular
systems. Jagarlamudi and Ahmad et al. used ultraviolet–visible spec-
troscopy to directly detect Au3+ produced by the reaction of AuTM and
ClO- in the PBS solution [8,9]. Jagarlamudi et al. used ion chromatog-
raphy to prove that the isolated MPO could yield Au3+ from AuTM by
producing HClO in the presence of Cl- and H2O2 [8]. Gleichmann et al.
* Corresponding authors at: Key Laboratory of Xinjiang Phytomedicine Resources of Ministry of Education, School of Pharmacy, Shihezi University, Shihezi
832002, China (S. Sun).
E-mail addresses: hcl_tea@shzu.edu.cn (C. Hong), sunsg@nwsuaf.edu.cn (S. Sun).
https://doi.org/10.1016/j.bioorg.2021.104749
Received 16 January 2021; Received in revised form 9 February 2021; Accepted 13 February 2021
Available online 19 February 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

Z. Xie et al.
Bioorganic Chemistry 110 (2021) 104749
Scheme 1. The synthetic route of RA.
used T lymphocytes to indirectly analyze Au3+ produced in AuTM-
treated mice [10,11]. Because Au3+ has a short lifespan in existence of
reducing agents, luminescence analysis method is indubitably the best
choice for monitoring of Au3+ in vivo owing to its real-time detection,
rapidity, visibility, high sensitivity and selectivity, and easy operation
[12,13].
2. Experimental section
2.1. Reagents
All organic reagents were attained from reagent vendors and are all
of analytical grade. Sodium aurothiomalate (99.9%) was obtained from
Alfa Aesar. 4-Aninobenzhydrazide (4-ABAH, 95%), lipopolysaccharide
(LPS) and MTT were bought from Solarbio. Gold (III) chloride hydrate
were purchased from Macklin.
The last few decades have witnessed the flourishing development of
luminescent probes which are designed for Au3+ ions detection [14–16].
To date, luminescent probes based on various fluorophore units
including rhodamine [17,18], fluorescein [16], BODIPY [19], naph-
thylamide [20], coumarins [21] and etc. have been employed success-
fully to detect Au3+ ions. However, organic probes have some
undesirable characteristics, such as Water insolubility, low photo-
stability, small Stokes shift and pH-dependent fluorescence. Because the
Ru(II)-complexes have eminent luminescence properties, photochemical
stabilities, intense visible excitation and emission, a large Stokes shift,
water solubility and a wide pH-availability range [22], they are counted
on to be the most widely used transition metal complexes as molecular
reporters. More importantly, ruthenium (II) compounds have the char-
acteristics of low toxicity, easy to absorb and rapid metabolism in vivo
[23–25]. So many luminescent probes/sensors using ruthenium (II)
complexes as molecular reports have been evolved for the qualitative
and quantitative analysis of anions, metal ions, molecular oxygen and
biological molecules in organisms. For example, Yuan et al. reported
synthetic Ru-MDB probes to monitor H2S in live leeches, zebrafish em-
bryos, adult zebrafish and mice by in-vivo imaging [26]. In previous
work, our group designed and synthesized a reversible selective lumi-
nescence probe based on a Ru(II)-complex in water solution for the
detection of Cu2+ and imaging of Cu2+ in live pea aphids [27], and
designed a dinuclear Ru(II)-complex Ruazo for detecting HClO and
imagined with flying colors in cells and mice [28]. Whereas, to our
knowledge, there is no report on the probe based on ruthenium (II)
2.2. Apparatus analysis
1H NMR and 13C NMR spectra were documented on a Bruker 500
AVANCE III spectrometer, chemical displacements expressed in ppm (1H
NMR 500 MHz, 13C NMR 125 MHz, Germany). Mass spectra data was
collected by Thermo Fisher LCQ Fleet mass spectrometer (USA). Ab-
sorption spectra were obtained by using a Shimadzu UV-1750 UV–Vis
spectrometer (Japan). Luminescence spectra data was gained with a
Shimadzu RF-5301 fluorescence spectrometer (Japan). The cell image
data was collected by confocal fluorescence microscopes (Axio Imager
A2, Zeiss; CLSM, Nikon A1R). Zebrafish imaging was performed on small
animal imaging system (Night OWL II LB 983 NC100, Berthold,
Germany).
2.3. Cytotoxicity assay of RA
All the cells used in the experiment were cultured in an incubator
with 5% CO2 at 37 ℃. HepG-2 cells were added in 96-well microplates at
a density of 1 × 105 cells/mL. After 24 h of cell attachment, cells were
cultured in medium with various concentrations (0, 2.5, 5, 10, 20 and
compound for monitoring Au3+
.
40 μM) of RA for 24 h. MTT was added to each well, and the plate was
Hence, we synthesized a novel water-soluble Ru (II) complex-based
luminescent probe (RA) for detecting Au3+(Scheme 2). Compared
with other probes, RA showed a wide pH-availability range, a large
Stokes shifts, and good water solubility and cell membrane permeability
(Table S1). In this work, our goals were to (a) synthesize a water-soluble
ruthenium compound-based luminescent probe suitable for monitoring
Au3+ in vivo and in vitro, (b) demonstrate the practicability of the probe
in cell and zebrafish, and (c) prove that the luminescent probe detection
technology can capture the production of Au3+, which is one of inter-
mediate metabolite of gold (I) drugs, in living cells and living bodies,
and provide a new method for studying the metabolism mechanism and
the development of gold (I) medicine and their applications.
placed in the incubator for 4 h. The absorbance was record at 490 nm by
microplate reader (KHB ST-360).
2.4. Luminescence detection of live cells and adult zebrafish with RA
HepG-2 cells were incubated for another 1 h with 20
μ
M RA. Then
the cells were treated with Au3+ (10
μM) respectively and incubated for
30 min for imaging. Adult zebrafish was cultured in six-well plates and
was incubated in RA solution (5 μM) for 30 min. Then the zebrafish
abdomen was separately injected by AuTM with various concentrations
(0, 0.1, 0.2, 0.4, 0.8, 1.6 mg/kg) for imaging (λex = 475 nm, λem = 600 ±
20 nm).
2

Z. Xie et al.
Bioorganic Chemistry 110 (2021) 104749
Scheme 2. Recognition mechanism of RA towards Au3+
.
Fig. 1. (a) Luminescent spectra of RA (20 µM) upon titration with Au3+ in PBS (pH 7.2). (b) The emission intensity of RA at 592 nm changes with the concentration
of Au3+. (c) The emission spectra of RA (20 µM) changes with various metal ions (4 eq.). (d) The luminescent intensity changes of RA with addition of various metal
ions and Au3+. Cyan bars represent different ions, Red bars represent the subsequent addition of Au3+ (2 eq.) to aforementioned solutions, λex = 468 nm, λem
=
592 nm.
2.5. Imaging of LPS-induced macrophages and adult zebrafish with AuTM
3. Results and discussion
RAW 264.7 cells were incubated in DMEM medium containing 10%
FBS, 1% penicillin/streptomycin. PMQ were obtained by Kunming mice
and Cells were obtained from Kunming mice, refer to literature for
specific operation methods [29]. The cells were added in a six-well plate
with 18-mm coverslips per well. The macrophages (RAW 264.7 and
3.1. Synthesis of probe RA and proposed mechanism
The synthetic route of probe RA is summarized in Scheme 1. Com-
pound 2 was prepared according to a Steck-Day reaction procedure [30].
The ligand (Compound 3) was prepared following a modified Duff re-
action based on compound 2 [31]. The Ru-complex RA was gained with
a considerable yield (85%) by direct reaction of the Compound 3 with
the suitable molar ratios of cis-[Ru(phen)2Cl2] in anhydrous ethanol,
which was confirmed by MS, 13C NMR and 1H NMR (Figure S1-S3a). The
detail synthesis can be found in the supporting information. The
recognition mechanism of RA to Au3+ can be reasonably explained by
the formation of coordination interaction between oxygen of RA and
Au3+ (Scheme 2) [32,20]. Job’s plot showed a maximum emission in-
tensity value at a molar fraction of 0.5, which gave a solid evidence for
the formation of 1:1 complex of RA-Au3+ (Fig. S6). The 1H NMR
PMQ) were pretreated in LPS (1 μ
g/mL) for 24 h at 37 ◦C. The cells pre-
incubated with 4-ABAH (MPO inhibitor) and then stimulated with LPS
for 24 h to ensure that metabolites formation was MPO dependent.
Then, the cells were treated further with AuTM (50 μM) for 1 h. Probe
RA was added and the cells were incubated for 1 h and imaged.
The abdomen of adult zebrafish was injected 1 mg/mL LPS to induce
inflammation for 24 h and then washed twice with PBS buffer AuTM (50
μ
M). Subsequently, zebrafish was incubated for 30 min with RA (5 μM).
Adult zebrafish imaging data was collected by the Night QWL (λex = 475
nm, λem = 600 ± 20 nm).
3

Z. Xie et al.
Bioorganic Chemistry 110 (2021) 104749
Fig. 2. CLSM images of HepG-2 cells incubated with RA (20
μ
M) for 1 h (a), followed by incubation with (a) and without (b) 10
μ
M Au3+ for 30 min. (c) Lumi-
nescence images of adult zebrafish incubated with RA (5
μ
M) for 30 min, followed by injection with Au3+ (0, 0.1, 0.2, 0.4, 0.8, 1.6 mg/kg).Scale bar: 20
μm.
spectrum (Fig. S3) provided some further evidence.
3.4. Cytotoxicity assay and cellular imaging
3.2. Luminescent properties of RA
To demonstrate the feasibility of RA in living cells and in vivo.
Firstly, the MTT method was used to detect the cytotoxicity of RA. As
shown in Fig. S8, even if the RA concentration reaches 40 µM, the cell
survival rate still exceeds 85% after 24 h, indicating that the probe was
almost nontoxic. Then, we used CLSM to perform live cell imaging to
The luminescent properties of RA towards Au3+ were studied.
Firstly, the probe RA responded well to Au3+ in the pH region of 3–8,
and physiological pH region (pH 7.2) was used for further study
(Fig. S7). The changes in the luminescence spectra of probe RA (20
μ
M)
assess the cell permeability of RA and the ability of RA to detect Au3+
.
with or without different concentrations of Au3+ (0–40
μ
M) in PBS (pH
As shown in Fig. 2b, without Au3+, free RA displayed strong lumines-
7.2) are showed in Fig. 1a. The emission intensity at 592 nm decreased
cence signals in living cells. In comparison, after addition of Au3+
,
gradually on addition of Au3+ with a good linear relationship between
decreased red luminescence was obviously observed (Fig. 2a). The data
demonstrated that RA exhibits good cell-membrane permeability and
could detect Au3+ in living cells.
the luminescence intensity and the Au3+ concentration from 0 to 40
μM,
as shown in Fig. 1b. The detection limit was calculated to be 135 nM.
According to the Benesi-Hildebrand Method [33], the binding constant
of RA with Au3+ in PBS solution was calculated to be 1.48 × 104 Mꢀ 1
.
3.5. Luminescence imaging of Au3+ in zebrafish
3.3. Selectivity of RA for Au3+
In the preliminary work, we designed a malondialdehyde-specific
detection probe, which was successfully imaged the adult zebrafish
[34]. Therefore, encouraged by preliminary work, we examined that RA
could visualize Au3+ in adult zebrafish. As shown in Fig. 2c, RA showed
a strong luminescence signal in the control group (0 mg/kg Au3+), and
with the increasing concentration of Au3+ solution injected to zebrafish,
the luminescence signal gradually weakened. The data demonstrated
that RA exhibits good biocompatibility and rapid responsiveness in
adult zebrafish, which could be used to detect Au3+ in vivo.
To assess the selective property of RA for Au3+, the effects of various
physiological active metal ions on RA were studied. Under identical
conditions, RA shows no luminescence change after the addition of any
of above-mentioned metal ions. However, when Au3+ was added into
the solutions of those metal ions with RA, a significant decrease of
luminescence emission at 592 nm was observed and the solution is
colorless from orange, which shows that the presence of other metal ions
has almost no impact on the detection of Au3+ (Fig. 1c and d). Therefore,
RA could selectively identify Au3+ ions in the solution with other
species.
3.6. Detection of intermediate metabolite of AuTM in RAW264.7 and
PMQ cells
In view of the advantages of RA probe in cells and zebrafish, we have
4

Z. Xie et al.
Bioorganic Chemistry 110 (2021) 104749
Fig. 3. (a) Visualization of metabolite Au3+ of different concentration AuTM in RAW264.7 cells. (0
μ
M, 10
μM, 20 μM, 50 μM and 100 μM), quantitative lumi-
nescence intensity using Image J Software; The viability of cells incubated in serum-free medium (b) and 1
μ
g/mL LPS (c) for 24 h, and then treated with AuTM (0, 5,
10, 20, 50, 100, 200, 500, 1000 μM) for 24 h.
Fig. 4. Application of RA (20 µM) for visualization of metabolite Au3+ of AuTM. Imaging of metabolite Au3+ of AuTM (50 µM) in RAW264.7 cells (a) and PMQ (b)
using a fluorescence microscope. Quantitative luminescence intensity of (c) and (d) using Image J Software. The results are presented as means ± SD (n = 3).
Concentration of LPS: 0.5 μg/mL (PMQ), 1.0 μg/mL (RAW264.7 cells).
entered further in-depth studies on the production of AuTM intermedi-
ate metabolites and their production mechanisms in vitro and in vivo. In
vitro studies show that HClO can oxidize Au+ in AuTM to Au3+, as show
in Fig. S9, it was found that the reaction between AuTM and HClO would
reduce the luminescence intensity of RA, indicating the generation of
Au3+. This result is in good agreement with the literature [35]. In order
to prove that AuTM produces Au3+ in complex cell system, LPS-induced
RAW264.7 cells were treated with virous concentrations of AuTM for 1
h, and performed luminescence imaging with RA. RA has good photo-
stability in LPS-treated RAW264.7 cells, as shown in Fig.S10. Whereas,
with the increase of the concentration of AuTM, the luminescence in-
tensity decreased gradually (Fig. 3a). More interestingly, we found that
There was an opposite trend of AuTM on the viability of induced-
RAW264.7 cells and normal RAW264.7 cells. As shown in Fig. 3b the
activity of RAW264.7 cells treated with different concentrations of
AuTM did not decrease or even slightly increased [36]. In comparison,
RAW264.7 cells were induced by LPS (1 μg/mL) and then treated with
different concentrations of AuTM, the cell viability decreased signifi-
cantly with the increase of AuTM concentration (Fig. 3c). The reason
may be that LPS induces MPO release in RAW264.7 cells to produce
5

Z. Xie et al.
Bioorganic Chemistry 110 (2021) 104749
Fig. 5. Luminescence imaging of Au3+ produced by AuTM in LPS-induced zebrafish. (a) Adult zebrafish injected with AuTM (10 mg/kg, 10
μL) 24 h after being
injected PBS and LPS (1 mg/mL, 10
5.0, 10.0 mg/kg, 10
μL) and then incubated with RA (5 μM) for 30 min. (b) Adult zebrafish injected different concentration AuTM (0, 0.5, 1.0, 2.0,
μ
L) by i.p. injection pre-treated with LPS (1 mg/mL, 10 μL) injected zebrafish for 24 h and then incubated with RA (5 μM) for 30 min.
HClO, and then HClO reacts with AuTM to produce Au3+, which leads to
the decrease of cell viability [37,38].
utility of RA for Au3+ detection has been fully verified in living cells and
adult zebrafish. The probe was able to monitor the intermediate me-
tabolites Au3+ of AuTM in LPS-induced RAW264.7 cells, PMQ and adult
zebrafish, and proved its produce mechanism. In view of the high
instability of drug intermediate metabolites in vivo, the luminescent
probe detection method with fast response and visualization is expected
to become a potential tool for drug metabolism detection.
Subsequently, we used 4-ABAH to inhibit the expression of MPO in
RAW264.7 and PMQ cells to verify that Au3+, the intermediate metab-
olite of AuTM, was produced by the HClO of the MPO-H2O2-halide
system in macrophages. Firstly, AuTM (50 μM) treated RAW264.7 cells,
after LPS induction, the luminescence of RAW264.7 cells decreased
(Fig. 4a, middle). Interestingly, treatment of cells with 4-ABAH (200
Author contributions
μ
M) resulted in the restoration of luminescence, confirming the oxida-
Zhenzhen Xie, Prof. Chenglin Hong and Prof. Shiguo Sun constructed
the idea and designed the whole assay. Zhenzhen Xie finished experi-
ments, analyzed data and wrote the paper. Jia Wen designed and syn-
thesized the probe and completed the relevant tests. Shaowei Sun
participated in the synthesis of the probe. Xiling Deng provided the
support of synthesis. Jing Zhang and Bo Zhang provided the support of
cell and zebrafish experiments. Shichao Han and Lixia Wang provided
scientific advice on establishment of cell and zebrafish model.
tion reaction between AuTM and endogenous HClO in cells to produce
intermediate Au3+ (Fig. 4a, right). Subsequently, in order to verify the
reliability of this result, we used the same method to treat PMQ cells, and
the results were consistent with the above results (Fig. 4b).
3.7. Luminescence imaging of LPS-induced and zebrafish treated with
AuTM
LPS administration to zebrafish has frequently been used as a model
for inflammation model and results in endogenous HClO production
[39]. In order to further verify the practicality of RA detection of AuTM
intermediate Au3+ in vivo, zebrafish were injected with AuTM solution
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
24 h after being injected without and with LPS (1 mg/mL, 10 μL). The
results showed that the control group had strong luminescence, whereas
the group injected LPS was showed decreased luminescence (Fig. 5a)
and even luminous intensity gradually decreases with increasing AuTM
concentration (Fig. 5b). These results confirmed that Au3+ was gener-
ated by AuTM in an inflammatory state.
Acknowledgments
This work was supported by the National Natural Science Foundation
of China [grant number U1803283, 21878249]; the Study on the Key
Technique Improvement of Xinjiang Licorice Planting and Quality
Control of Xinjiang Production and Construction Corps [grant number
2018AB012]; Graduate Research Innovation Project of Xinjiang [grant
number XJ2020G128].
3.8. Advantages of the method
Compared with the method mentioned in Table S2, the luminescence
detection method can not only directly detect Au3+, the intermediate
metabolite of AuTM, but also realize real-time visualized in-situ detec-
tion in RAW264.7 cells, PMQ and adult zebrafish. By using conventional
physicochemical methods, such as UV–vis spectroscopy and Ion Chro-
matography, but it is difficult to directly demonstrate Au3+ in the
presence of whole cells. Since Au3+ will quickly be returned to Au+ with
the coexistence of thiol reducing agent, Au3+ exists for a short time in
cells. Due to the process of Au3+ being reduced to Au+ leaves behind
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.104749.
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